Lingjuan Liu

Inhibition of Histone H3K9 Acetylation by Anacardic Acid Can Correct the Over-Expression of Gata4 in the Hearts of Fetal Mice Exposed to Alcohol during Pregnancy

Background Cardiovascular malformations can be caused by abnormalities in Gata4 expression during fetal development. In a previous study, we demonstrated that ethanol exposure could lead to histone hyperacetylation and Gata4 over-expression in fetal mouse hearts. However, the potential mechanisms of histone hyperacetylation and Gata4 over-expression induced by ethanol remain unclear. Methods and Results Pregnant mice were gavaged with ethanol or saline. Fetal mouse hearts were collected for analysis. The results of ethanol fed groups showed that global HAT activity was unusually high in the hearts of fetal mice while global HDAC activity remained unchanged. Binding of P300, CBP, PCAF, SRC1, but not GCN5, were increased on the Gata4 promoter relative to the saline treated group. Increased acetylation of H3K9 and increased mRNA expression of Gata4, α-MHC, cTnT were observed in these hearts. Treatment with the pan-histone acetylase inhibitor, anacardic acid, reduced the binding of P300, PCAF to the Gata4 promoter and reversed H3K9 hyperacetylation in the presence of ethanol. Interestingly, anacardic acid attenuated over-expression of Gata4, α-MHC and cTnT in fetal mouse hearts exposed to ethanol. Conclusions Our results suggest that P300 and PCAF may be critical regulatory factors that mediate Gata4 over-expression induced by ethanol exposure. Alternatively, P300, PCAF and Gata4 may coordinate over-expression of cardiac downstream genes in mouse hearts exposed to ethanol. Anacardic acid may thus protect against ethanol-induced Gata4, α-MHC, cTnT over-expression by inhibiting the binding of P300 and PCAF to the promoter region of these genes.

p300-Mediated Histone Acetylation is Essential for the Regulation of GATA4 and MEF2C by BMP2 in H9c2 Cells

Histone acetylase (HAT) p300 plays an important role in the regulation of cardiac gene expression. During cardiac development, bone morphogenetic protein (BMP)-2 induces the expression of cardiac transcription factors. However, the underlying molecular mechanism(s) is not clear. In the present study, we tested the hypothesis that p300-mediated histone acetylation was essential for the regulation of cardiac transcription factors by BMP2. Cultured rat H9c2 embryonic cardiac myocytes (H9c2 cells) were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without curcumin, a specific p300-HAT inhibitor. Quantitative real-time RT-PCR analysis showed that curcumin substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and MEF2C, but not Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that curcumin inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and MEF2C, but not of Tbx5. In addition, curcumin selectively suppressed AdBMP2-induced expression of HAT p300, but not HAT GCN5 in H9c2 cells. The data indicated that inhibition of histone H3 acetylation with curcumin diminished BMP2-induced expression of GATA4 and MEF2C, suggesting that p300-mediated histone acetylation was essential for the regulation of GATA4 and MEF2C by BMP2 in H9c2 cells.

Inhibition of histone acetylation by curcumin reduces alcohol-induced fetal cardiac apoptosis.

BackgroundPrenatal alcohol exposure may cause cardiac development defects, however, the underlying mechanisms are not yet clear. In the present study we have investigated the roles of histone modification by curcumin on alcohol induced fetal cardiac abnormalities during the development.Methods and resultsQ-PCR and Western blot results showed that alcohol exposure increased gene and active forms of caspase-3 and caspase-8, while decreased gene and protein of bcl-2. ChIP assay results showed that, alcohol exposure increased the acetylation of histone H3K9 near the promoter region of caspase-3 and caspase-8, and decreased the acetylation of histone H3K9 near the promoter region of bcl-2. TUNEL assay data revealed that alcohol exposure increased the apoptosis levels in the embryonic hearts. In vitro experiments demonstrated that curcumin treatment could reverse the up-regulation of active forms of caspase-3 and caspase-8, and down-regulation of bcl-2 induced by alcohol treatment. In addition, curcumin also corrected the high level of histone H3K9 acetylation induced by alcohol. Moreover, the high apoptosis level induced by alcohol was reversed after curcumin treatment in cardiac cells.ConclusionsThese findings indicate that histone modification may play an important role in mediating alcohol induced fetal cardiac apoptosis, possibly through the up-regulation of H3K9 acetylation near the promoter regions of apoptotic genes. Curcumin treatment may correct alcohol-mediated fetal cardiac apoptosis, suggesting that curcumin may play a protective role against alcohol abuse caused cardiac damage during pregnancy.

Differentiation of mesenchymal stem cells into cardiomyocytes is regulated by miRNA-1-2 via WNT signaling pathway

BackgroundBone marrow derived stem cells (BMSCs) have the potential to differentiate into cardiomyocytes, but the rate of differentiation is low and the mechanism of differentiation is unclear completely. Here, we aimed to investigate the role of miR1-2 in differentiation of mouse BMSCs into cardiomyocyte-like cells and reveal the involved signaling pathways in the procedure.MethodsMouse BMSCs were treated with miR1-2 and 5-azacytine (5-aza). The expression of cardiac cell markers: NKx2.5, cTnI and GATA4 in BMSCs were examined by qPCR. The apoptosis rate was detected by flow cytometry and the activity of the Wnt/β-catenin signaling pathway was evaluated by measuring the upstream protein of this signaling pathway.ResultsAfter over-expression of miR1-2 in mouse BMSCs, the apoptosis rate was significantly lower than the 5-aza group, while the expressions of cardiac-specific genes: such as Nkx2.5, cTnI and GATA4 were significantly increased compared to the control group and the 5-aza group. Meanwhile, over-expression of miR1-2 in mouse BMSCs enhanced the expression of wnt11, JNK, β-catenin and TCF in the Wnt/β-catenin signaling pathway. Use of LGK-974, an inhibitor of Wnt/β-catenin signaling pathway, significantly reduced the expression of cardiac-specific genes and partially blocked the role of the miR1-2.ConclusionOver-expression of miR1-2 in mouse BMSCs can induce them toward promoted cardiomyocyte differentiation via the activation of the Wnt/β-catenin signaling pathway. Compared to 5-aza, miR1-2 can induce differentiation of BMSCs into cardiomyocytes more effectively with a less cytotoxicity.

Prenatal alcohol exposure causes the over-expression of DHAND and EHAND by increasing histone H3K14 acetylation in C57 BL/6 mice.

Prenatal alcohol exposure leads to congenital heart abnormal development, its mechanisms are still unknown. Recent reports have associated alcohol exposure with histone H3 acetylation. In the present study, we have performed the experiments to test the hypothesis that histone H3K14 acetylation is the key role in the fetal heart leads to over-expression of cardiac specific genes DHAND and EHAND caused by prenatal alcohol exposure. Seventy pregnant C57BL/6 mice were divided randomly into seven groups (n=10). They were the untreated group, dimethyl sulfoxide group, alcohol exposure group, curcumin treatment group, both alcohol and curcumin treatment group, SAHA treatment group, both alcohol and SAHA treatment group. Fetal mouse hearts were collected on embryonic day 14.5. The changes of HATs activities, the acetylation levels of histone H3K14 (H3K14ac), the expression levels of cardiac specific genes DHAND and EHAND, and structure of chromatin were determined. Our data indicates that curcumin and SAHA significantly reduces and increases the activities of HATs and the levels of histone H3K14ac in fetal hearts, respectively. The expression of DHAND and EHAND is significantly down-regulated and up-regulated in the groups treated with curcumin and SAHA. Furthermore, our results from ChIP assays have shown that the histone H3K14ac connects with the DHAND and EHAND genes are significantly inhibited by curcumin and simulated by SAHA. Our study suggests that prenatal alcohol exposure causes the over-expression of DHAND and EHAND by increasing H3K14ac in mice.

Alcohol exposure increases the expression of cardiac transcription factors through ERK1/2-mediated histone3 hyperacetylation in H9c2 cells

Alcohol abuse during pregnancy may cause fetal cardiac developmental abnormalities. Our previous studies showed that alcohol could induce histone hyperacetylation and over-expression of cardiac transcription factors both in vivo and in vitro. The objective of the present study was to investigate the role of ERK1/2 signaling pathway in alcohol-induced histone hyperacetylation and up-regulation of cardiac transcription factors in H9c2 cells. The Cardiac cell line H9c2 was cultured with alcohol. U0126, a specific inhibitor of ERK1/2 pathway was employed to block the ERK1/2 signaling pathway. Western blotting analysis showed that alcohol significantly enhanced the levels of phosphorylated ERK1/2 and induced hyperacetylation of histone3, which were both effectively prevented with U0126. Real-time PCR showed that U0126 treatment significantly decreased alcohol-induced over-expression of GATA4 and MEF2c, and the basal expression level of GATA4, but did not affect MEF2c. ChIP assay showed that U0126 treatment significantly decreased alcohol-induced hyperacetylation of histone3 near the promoter regions of GATA4 and MEF2c. The basal acetylation level of histone3 near the promoter region of GATA4 was affected by U0126 as well, but not that near the promoter region of MEF2c. These data indicated that ERK1/2 signaling played an important role in mediating alcohol induced over-expression of GATA4 and MEF2c, which is possibly through the up-regulation of acetylation of histone3 near the gene promoters that affects the expression of GATA4 and MEF2c in H9c2 cells. ERK1/2 pathway might be a potential target for the intervention of alcohol induced congenital heart diseases.

Measurements in Pediatric Patients with Cardiomyopathies: Comparison of Cardiac Magnetic Resonance Imaging and Echocardiography.

Aims: Cardiomyopathies are common cardiovascular diseases in children. Cardiac magnetic resonance imaging (cMRI) and echocardiography (Echo) are routinely applied in the detection and diagnosis of pediatric cardiomyopathies. In this study, we compared and explored the correlation between these two measurements in pediatric patients with various cardiomyopathies. Methods and Results: A total of 53 pediatric patients with cardiomyopathy hospitalized during the recent 3 years in our hospital were analyzed. All of them and 22 normal controls were assessed by both cMRI and Echo. Cardiac function of the patients was graded according to the New York Heart Association functional classification. The cardiac function indexes measured with both cMRI and Echo included left-ventricular (LV) end-diastolic volume (EDV), end-systolic volume, ejection fraction and fractional shortening. These parameters were somehow lower in cMRI measurements than in Echo measurements. The index of diastolic function, such as peak filling rate (PFR) measured with cMRI, had a good correlation with the clinical cardiac functional score, while the index of the diastolic function (early/atrial filling ratio and isovolumic relaxation time) measured with Echo was not well correlated with the clinical cardiac function score. Significant systolic dysfunction was detected by cMRI in 34 patients with dilated cardiomyopathy, LV noncompaction or endocardial fibroelastosis. Significant diastolic dysfunction was detected by cMRI in 19 patients with hypertrophic cardiomyopathy or restrictive cardiomyopathy showing an alteration in PFR and EDV. Conclusion: Both cMRI and Echo are of great value in the diagnosis and assessment of cardiac function in pediatric patients with cardiomyopathy. cMRI could accurately display the characteristic morphological changes in the hearts affected with cardiomyopathies, and late gadolinium enhancement on cMRI may reveal myocardial fibrosis, which has obvious advantages over Echo measurements in diagnosis. Furthermore, cMRI can quantitatively determine ventricular function because it does not make invalid geometrical assumptions.

Epigallocatechin gallate reverses cTnI‐low expression‐induced age‐related heart diastolic dysfunction through histone acetylation modification

Cardiac diastolic dysfunction (CDD) is the most common form of cardiovascular disorders, especially in elderly people. Cardiac troponin I (cTnI) plays a critical role in the regulation of cardiac function, especially diastolic function. Our previous studies showed that cTnI‐low expression induced by histone acetylation modification might be one of the causes that result in diastolic dysfunction in ageing hearts. This study was designed to investigate whether epigallocatechin‐3‐gallate (EGCG) would modify histone acetylation events to regulate cTnI expression and then improve cardiac functions in ageing mice. Our study shows that EGCG improved cardiac diastolic function of aged mice after 8‐week treatment. Low expression of cTnI in the ageing hearts was reversed through EGCG treatment. EGCG inhibited the expression of histone deacetylase 1 (HDAC1) and HDAC3, and the binding levels of HDAC1 in the proximal promoter of cTnI. Acetylated lysine 9 on histone H3 (AcH3K9) levels of cTnIs promoter were increased through EGCG treatment. Additionally, EGCG resulted in an ascent of the binding levels of transcription factors GATA4 and Mef2c with cTnIs promoter. Together, our data indicate that EGCG may improve cardiac diastolic function of ageing mice through up‐regulating cTnI by histone acetylation modification. These findings provide new insights into histone acetylation mechanisms of EGCG treatment that may contribute to the prevention of CDD in ageing populations.

Diastolic dysfunction and cardiac troponin I decrease in aging hearts.

Cardiac tropnoin I (cTnI) plays a critical role in the regulation of diastolic function, and its low expression may result in cardiac diastolic dysfunction, which is the most common form of cardiovascular disorders in older adults. In this study, cTnI expression levels were determined in mice at various ages and cardiac function was measured and compared between young adult mice (3 and 10 months) and older mice (18 months). The data indicated that the cTnI levels reached a peak high in young adult hearts (3 months), but decreased in older hearts (18 months). Furthermore, the older hearts showed a significant diastolic dysfunction observed by P-V loop and echocardiography measurements. To further define the mechanism underlying the cTnI decrease in aging hearts, we tested DNA methylation and histone acetylation modifications of cTnI gene. We found that acetylation of histone near the promoter region of cTnI gene played an important role in regulation of cTnI expression in the heart at different ages. Our study indicates that epigenetic modification caused cTnI expression decrease is one of the possible causes that result in a reduced cTnI level and diastolic dysfunction in the older hearts.

Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.